Autoantibodies To Neutrophil Cytoplasmic Antigens Research Articles

Twenty-five years of European Union collaboration in ANCA-associated vasculitis research.

This special edition reviews the progress in understanding of systemic vasculitis associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). European research groups have contributed to this research including the original observations of the association between ANCA and vasculitis. Areas of interest include classification and epidemiology, genetics and pathogenesis, disease assessment, histology and long-term outcomes. Clinical trials conducted by the European Vasculitis Study group have helped to define the current standard of care for the treatment of patients with vasculitis and provided a platform for the investigation of newer therapies. The prognosis of patients with ANCA-associated vasculitis has improved over this period as a result of facilitated diagnosis and development of consensus, evidence-based, treatment recommendations. The ANCA story represents an example of the power of a biomarker in influencing a disease area, inspiring research and providing physicians with better tools to treat patients with these disorders.

Azathioprine Hypersensitivity Presenting as Cardiogenic Shock and Sweet's Syndrome in a Patient with Microscopic Polyangiitis

Azathioprine hypersensitivity is a clinical syndrome which may manifest from isolated fever and rash to multi-organ failure. This rare condition is usually self-limiting following the discontinuation of azathioprine. Therefore, it is important to maintain a high index of clinical suspicion for hypersensitivity reactions with azathioprine therapy. We report a case of azathioprine hypersensitivity in a 69-year-old woman who developed cardiogenic shock and Sweet's syndrome following the initiation of azathioprine for her underlying autoantibodies to neutrophil cytoplasmic antigens (ANCA) associated microscopic polyangiitis.

Molecular mimicry in pauci-immune focal necrotizing glomerulonephritis

Pauci-immune focal necrotizing glomerulonephritis (FNGN) is a severe inflammatory disease associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA). Here we characterize autoantibodies to lysosomal membrane protein-2 (LAMP-2) and show that they are a new ANCA subtype present in almost all individuals with FNGN. Consequently, its prevalence is nearly twice that of the classical ANCAs that recognize myeloperoxidase or proteinase-3. Furthermore, antibodies to LAMP-2 cause pauci-immune FNGN when injected into rats, and a monoclonal antibody to human LAMP-2 (H4B4) induces apoptosis of human microvascular endothelium in vitro. The autoantibodies in individuals with pauci-immune FNGN commonly recognize a human LAMP-2 epitope (designated P(41-49)) with 100% homology to the bacterial adhesin FimH, with which they cross-react. Rats immunized with FimH develop pauci-immune FNGN and also develop antibodies to rat and human LAMP-2. Finally, we show that infections with fimbriated pathogens are common before the onset of FNGN. Thus, FimH-triggered autoimmunity to LAMP-2 provides a previously undescribed clinically relevant molecular mechanism for the development of pauci-immune FNGN.

Randomized Trial of Plasma Exchange or High-Dosage Methylprednisolone as Adjunctive Therapy for Severe Renal Vasculitis

Systemic vasculitis associated with autoantibodies to neutrophil cytoplasmic antigens (ANCA) is the most frequent cause of rapidly progressive glomerulonephritis. Renal failure at presentation carries an increased risk for ESRD and death despite immunosuppressive therapy. This study investigated whether the addition of plasma exchange was more effective than intravenous methylprednisolone in the achievement of renal recovery in those who presented with a serum creatinine >500 micromol/L (5.8 mg/dl). A total of 137 patients with a new diagnosis of ANCA-associated systemic vasculitis confirmed by renal biopsy and serum creatinine >500 micromol/L (5.8 mg/dl) were randomly assigned to receive seven plasma exchanges (n = 70) or 3000 mg of intravenous methylprednisolone (n = 67). Both groups received oral cyclophosphamide and oral prednisolone. The primary end point was dialysis independence at 3 mo. Secondary end points included renal and patient survival at 1 yr and severe adverse event rates. At 3 mo, 33 (49%) of 67 after intravenous methylprednisolone compared with 48 (69%) or 70 after plasma exchange were alive and independent of dialysis (95% confidence interval for the difference 18 to 35%; P = 0.02). As compared with intravenous methylprednisolone, plasma exchange was associated with a reduction in risk for progression to ESRD of 24% (95% confidence interval 6.1 to 41%), from 43 to 19%, at 12 mo. Patient survival and severe adverse event rates at 1 yr were 51 (76%) of 67 and 32 of 67 (48%) in the intravenous methylprednisolone group and 51 (73%) of 70 and 35 of (50%) 70 in the plasma exchange group, respectively. Plasma exchange increased the rate of renal recovery in ANCA-associated systemic vasculitis that presented with renal failure when compared with intravenous methylprednisolone. Patient survival and severe adverse event rates were similar in both groups.

Autoantibodies to neutrophil cytoplasmic antigens (ANCA) do not bind to polymorphonuclear neutrophils in blood

Autoantibodies to neutrophil cytoplasmic antigens (ANCA), particularly to proteinase 3 (PR3), are found in the majority of patients with systemic Wegener's granulomatosis. The autoantibodies are widely used as diagnostic markers. Their role in the development and progression of the disease, however, is still under investigation. The primary target of ANCA, PR3, is located in the cytoplasm of polymorphonuclear neutrophils (PMN) or monocytes and is translocated to the cell surface upon stimulation. In patients with Wegener's granulomatosis PR3 is up-regulated most prominently during active disease. Despite the fact that both autoantibodies to PR3 and PMN expressing PR3 are present in patients with Wegener's granulomatosis, there is no evidence for binding of the autoantibodies to PMN. The present study was designed to analyze binding characteristics of autoantibodies to PR3 on PMN. PMN of patients with active Wegener's granulomatosis (N= 10) were tested for autoantibody binding. Despite high autoantibody titer and PR3 expression on the PMN, no surface-bound IgG was found on PMN ex vivo. When ANCA-containing plasma from patients was incubated with isolated PMN, stimulated to express PR3, again no specific binding of the autoantibody could be detected. Also keeping the samples on ice did not allow surface detection of IgG, ruling out degradation or internalization of the autoantibodies. Only when purified IgG fractions were used, binding to PMN was seen in 14 of 25 patients. Already 1% of plasma, however, was sufficient to greatly reduce the IgG binding. Reduced binding of the IgG fraction was also seen when a larger reaction volume was used. Our data indicate that autoantibodies to PR3 have a rather low affinity for surface-associated PR3 on PMN. This, in turn, argues against the hypothesis that ANCA contributes to the pathogenesis of the disease by stimulating viable PMN in whole blood.

Pulmonary-Renal Syndrome

Pulmonary-renal syndrome describes a dramatic presentation of multi-system autoimmune disease characterized by diffuse pulmonary hemorrhage and glomerulonephritis. Autoantibodies to neutrophil cytoplasmic antigens (ANCA) associated systemic vasculitis and anti-glomerular basement membrane (GBM) disease account for over 60 and 20%, respectively, and the presence of circulating ANCA or anti-GBM antibodies or both, provides a tool for diagnosis, classification and monitoring of disease. The close relation between ANCA and pauci-immune alveolar and glomerular capillaritis indicate a pathogenic role for these autoantibodies, further supported by evidence from experimental models. Respiratory failure due to pulmonary hemorrhage is the most frequent vasculitic cause of referral to an intensive care unit, where the course is often complicated and prolonged; it carries a mortality of 50% due to refractory hypoxia or secondary infection, and many survivors progress to end-stage renal failure. Aggressive treatment with corticosteroids, cyclophosphamide and plasma exchange can induce sustained remission, but success is dependent on rapid diagnosis, facilitated by the use of serological markers, careful monitoring of therapy, and avoidance of infection.

IgG Subclass Distribution and Complement Activation Ability of Autoantibodies to Neutrophil Cytoplasmic Antigens (ANCA)

To study the IgG subclass distribution and complement activation ability of ANCA, 24 sera containing C-ANCA (cytoplasmatic) and 7 sera containing P-ANCA (perinuclear), as determined by a routine immunofluorescence test, were examined. The subclass distribution was tested by the use of immunofluorescence and ELISA technique and monoclonal antibodies to IgG subclasses. The complement activating activity was studied by the use of immunofluorescence technique and antibodies to C3c and the terminal complement complex (TCC, C5b-9), directed against a neoepitope on C9. For C-ANCA, IgG1 and IgG4 were the dominating subclasses. The subclass distribution differed from that of other autoantibodies tested and that of the total IgG subclass concentrations. For P-ANCA, the results were inconclusive, but the lack of IgG3 was striking. All of the C-ANCA-containing sera caused deposition of C3c, and 50% of the sera gave formation of TCC when reacting with ethanol-fixed granulocytes. P-ANCA-containing sera caused some C3c deposition, but no TCC formation. The unusual IgG subclass distribution for C-ANCA is possibly due to repeated antigenic stimulations and/or to T cell factors influencing the antibody isotype switching. Despite the high IgG4 activity, sera containing C-ANCA are often able to activate complement and are therefore potentially harmful.

Serum soluble interleukin 2 receptor levels in anti-neutrophil cytoplasmic autoantibodies--positive systemic vasculitis

Systemic vasculitis is characterized by the presence of autoantibodies to neutrophil cytoplasmic antigens (ANCA). The role of T-lymphocytes in systemic vasculitis remains uncertain. In the present study, we attempted to explore the role of T-lymphocytes in systemic vasculitis by measuring the serum soluble interleukin 2 receptor (sIL2R) levels in seven vasculitic patients and comparing the sequential measurements with the titres of ANCA which satisfactorily reflect the disease activity. The serum levels of both ANCA and sIL2R were elevated at clinical presentation. Contrary to ANCA, the serum sIL2R remained elevated in most patients despite clinical remission following immunosuppressive therapy. These findings suggest that T-lymphocytes may be activated in the acute phase of the disease. The finding of elevated serum sIL2R levels in most patients during clinical remission indicates that it is not a good measure of the disease activity and tends to argue against the role of T-lymphocytes as a major effector mediating inflammatory injuries in systemic vasculitis.

Intravenous immunoglobulin in the therapy of systemic vasculitis

The current therapy of the systemic vasculitides with corticosteroid and cytotoxic drugs is successful in controlling disease in the majority of patients but is limited by adverse effects; drugs must also be continued in the long-term as relapse frequently follows their withdrawal. Autoantibodies to neutrophil cytoplasmic antigens (ANCA) are associated with systemic vasculitis and have been implicated in its pathogenesis. Interest in the use of pooled immunoglobulin (IVIg) in the therapy of vasculitis has followed the detection of antiidiotype antibodies to ANCA in IVIg and the success of IVIg in the treatment of Kawasaki disease, an ANCA positive childhood vasculitis. We have given IVIg to 12 patients with vasculitis, nine resistant to standard therapy, which led to clinical remission in eight and a partial response in four. Benefit was sustained in eleven although two had late relapses at 6 and 9 months. Clinical improvement was matched by falls in ANCA and C-reactive protein. The possible mechanisms underlying the therapeutic effect of IVIg in vasculitis are discussed.

T and B cell responses to neutrophil cytoplasmic antigens in systemic vasculitis

The systemic vasculitides (SV) are characterized by the presence of autoantibodies to neutrophil cytoplasmic antigens (ANCA). The role of T cells in SV is uncertain. We studied human and murine T cell responses to human neutrophil cytoplasmic antigens in vitro. T cells from mice immunized with the neutrophil extract showed dose-dependent antigen-specific proliferation, restricted by the MHC class II E molecule. Myeloperoxidase (MPO) was not an important target antigen for murine T cells. Peripheral blood lymphocytes (PBLs) were obtained from 36 patients with SV, 31 before the start of immunosuppressive therapy, and from 11 healthy controls. T cell responses to the neutrophil extract in vitro did not differ between patients and controls: there were only low levels of antigen-specific proliferation, and this could not be amplified by in vitro selection. In 3 patients and 2 normals, PBLs were also tested after the depletion of CD8+ cells; this did not unmask T cell reactivity to neutrophil extract. The lack of demonstrable T cell reactivity to this antigen preparation may indicate that T cells do not play an important effector role in these diseases. A solid-phase spot ELISA was adapted to demonstrate autoantibody-producing B cells in vitro. Low numbers of ANCA-producing B cells could be demonstrated in the majority of patients. B cells producing antibody to MPO could be demonstrated in most patients and in three laboratory staff, but not in normals from outside the laboratory. In 2 patients, sequential B cell spot ELISAs were performed during the introduction of therapy, and autoantibody-producing B cells rapidly decreased in number. This assay may therefore be useful in monitoring the effects of treatment at the cellular level.

Autoimmunity to lysosomal enzymes: new clues to vasculitis and glomerulonephritis?

The demonstration of autoantibodies to neutrophil cytoplasmic antigens (ANCA) in (1) systemic vasculitides such as Wegener's granulomatosis, classic polyarteritis nodosa and the Churg Strauss syndrome and (2) idiopathic crescentic glomerulonephritis has placed these disorders within the spectrum of autoimmune diseases. The target antigens have been identified as myeloid lysosomal enzymes that are involved in tissue destruction by neutrophils. Recent data suggest a pathogenetic role for these autoimmune responses. The autoantibodies may activate neutrophils, resulting in extensive tissue necrosis. Alternatively, T-cell-mediated immunity to these positively-charged enzymes that localize to polyamonic basement membranes may, in concert with the presence of autoantibodies, result in glomerulonephritis and vasculitis.